Cloning and characterization of phage in Vibrio parahaemolyticus

• Main Authors: Shin-Feng Lan, 藍世峰
• Other Authors: Hin-Chung Wong
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/06144070123996396442

碩士 === 東吳大學 === 微生物學系 === 92 === Vibrio parahaemolyticus is one of the most important foodborne pathogens in Taiwan. Since 1996, gastroenteritis caused by a specific serotype, new O3:K6, of V. parahaemolyticus has been widespread in Southeast Asia, East Asia including Japan, and North America. In Vibrio cholerae, the virulence genes are contained within the genome of CTXΦ, an integrated phage identified in 1996. We aimed to investigate the role of lysogenic phage in the new O3:K6 strains of V. parahaemolyticus. In our experiment, new O3:K6 with was treated with mitomycin C and two polyhedron phages, VP882 and VP1092, were identified. These phages showed a polyhedral head and a long tail which were associated with a special sheath-like protein. By polymerase chain reaction using primers specifically designed for these two phages, no homologous fragment of VP882 and VP1092 was found in other strains. Pulsed-field Gel Electrophoresis(PFGE) of two phage DNA revealed the genomes of VP882 and VP1092 were 38 and 46 Kbp, respectively. VP882 was further investigated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) of VP882 viron proteins showed two major polypeptides, 50KDa and 21KDa. Furthermore, five fragments of VP882 gemone were sequenced, namely, 13,880, 8,349, 2,283, 2, 081 and 2,983bp. The predicted amino sequences were similar to viral proteins found in GeneBank database.VP882 had open reading frames closely related to those of Vibrio harveyi bacteriophage VHML, another with 47% identity with the Vibrio vulnificus LuxR DNA-binding-domain. Roles of these phages in the phenotypes and virulence of these new O3:K6 strains have yet to be examined.